The development and homeostasis of the skeletal system depend on a higher-order network that ensures efficient balance between bone formation and resorption, which are effected by osteoblasts and osteoclasts, respectively. Perturbations of such a network are often associated with skeletal disorders, with the most severe being osteoporosis, with inherent risk of fracture and concomitant morbidity and mortality [1]. A master regulator of bone formation is the canonical Wnt signaling pathway, which exerts a tight control over several aspects of osteoblast differentiation and function [2-5]. The homeodomain protein TGIF functions as a transcriptional corepressor in multiple biological contexts, although its physiological role remains unclear [20, 21]. To advance our understanding of how TGIF's physiological functions and regulation are controlled, we have screened for TGIF-binding proteins using the yeast two-hybrid assay. Two previously unidentified TGIF binding proteins that attracted our attention are Axin2 and B56d (a B subunit of the phosphatase PP2A), both of which are operating as negative regulators of Wnt signaling through their potential to stabilize the destructosome, a signaling module where the protein kinase GSK3-[unreadable] marks [unreadable]-catenin for ubiquitin-dependent degradation [2, 22-26]. In subsequent gain- or loss-of- function studies, we found that TGIF contributes to the ability of Wnt to mediate transcriptional responses. We also obtained preliminary data demonstrating that TGIF is essential to the ability of Wnt signaling to induce osteoblast differentiation, revealing that one physiological function of TGIF is to ensure proper osteoblast cell fate determination in response to Wnt signaling. In support to this notion, we found that targeted disruption of TGIF in mice leads to impaired maturation of osteoblasts and decreased bone formation. Taken together, these findings led us to propose a working hypothesis in which TGIF may be involved in contributing to the regulation of Wnt signaling in bone, imposing stringent control over several components of canonical Wnt signaling pathway. Accordingly, the specific aims of this research proposal are: Aim 1: Further delineate the molecular mechanisms by which TGIF affects Wnt-mediated osteoblast differentiation and bone formation, with particular emphasis on its possible role in the assembly/disassembly of the destructosome complex, and thereby [unreadable]-Catenin stability. Aim 2: Analyze the effects of full and osteoblast-targeted conditional deletion of TGIF on bone homeostasis and the ability of Wnt to induce osteoblast differentiation and bone formation in vivo and in vitro. Functional characterization of TGIF as a bona fide contributor to the regulation of Wnt signaling and bone formation, will strengthen our knowledge of key physiological processes that maintain bone homeostasis, ultimately evolving into new concepts in the design and implementation of anabolic therapeutic drugs against osteoporosis and other low bone mass syndromes. PUBLIC HEALTH RELEVANCE: Wnt signaling fosters osteoblast proliferation and differentiation, and perturbations of this signaling pathway affect bone homeostasis, resulting in bone disorders in human. We discovered that one physiological function of the homeodomain protein TGIF is to ensure proper osteoblast cell fate determination in response to Wnt signaling. We believe that functional characterization of his newly discovered regulator of bone formation will open a new field of investigation both in terms of understanding the mechanisms that regulate osteoblasts and their bone matrix-secreting activity and in terms of drug discovery with the hope to improve the treatment of osteoporosis and other low bone mass syndromes.